N,N-dimethylacetamide targets neuroinflammation in Alzheimer’s disease in in-vitro and ex-vivo models

Alzheimer’s disease (AD) is a chronic degenerative brain disorder with no clear pathogenesis or effective cure, accounting for 60–80% of cases of dementia. In recent years, the importance of neuroinflammation in the pathogenesis of AD and other neurodegenerative disorders has come into focus. Previously, we made the serendipitous discovery that the widely used drug excipient N,N-dimethylacetamide (DMA) attenuates endotoxin-induced inflammatory responses in vivo. In the current work, we investigate the effect of DMA on neuroinflammation and its mechanism of action in in-vitro and ex-vivo models of AD. We show that DMA significantly suppresses the production of inflammatory mediators, such as reactive oxygen species (ROS), nitric oxide (NO) and various cytokines and chemokines, as well as amyloid-β (Aβ), in cultured microglia and organotypic hippocampal slices induced by lipopolysaccharide (LPS). We also demonstrate that DMA inhibits Aβ-induced inflammation. Finally, we show that the mechanism of DMA’s effect on neuroinflammation is inhibition of the nuclear factor kappa-B (NF-κB) signaling pathway and we show how DMA dismantles the positive feedback loop between NF-κB and Aβ synthesis. Taken together, our findings suggest that DMA, a generally regarded as safe compound that crosses the blood brain barrier, should be further investigated as a potential therapy for Alzheimer’s disease and neuroinflammatory disorders.

Preparation of Aβ42 oligomers. Aβ42 peptide (Invitrogen) was prepared as per the manufacturer's protocol. Briefly, 1 mg of Aβ42 was dissolved in 1 ml of 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP) (Sigma-Aldrich) and incubated for 1 h. After incubation, the HFIP was removed and dimethyl sulfoxide (DMSO) (ATCC) was added to resolve Aβ42 at a 1 mg/ml concentration. The solution was diluted to 100 μM with Minimum Essential Medium (MEM) (Corning) and incubated at 4 °C for 24 h. After 24-h incubation, the solution was centrifuged at 4 °C and 16,000×g for 10 min. The supernatant containing Aβ42 oligomers was used for experiments following established protocols 42,43 . Cell viability assay. HMC3 and SIM-A9 cells were seeded at 20,000 and 25,000 cells/well, respectively, in 96-well plates. After overnight incubation, the cells were incubated in the absence or presence of incremental concentrations (0.1-100 mM) of DMA for 2 h. To mimic experimental conditions, in which cells are stimulated with either LPS or Aβ, HMC3 cells and SIM-A9 cells were incubated with 1 μg/mL LPS (Escherichia coli 026:B6) (Sigma-Aldrich) or 3 μM Aβ42 for an additional 24 h, respectively. The concentration of 1 μg/mL LPS was chosen based on our long experience with eliciting cytokine secretion from cultured cells with this serotype of LPS 29 . The concentration of 3 μM Aβ42 was chosen based on reports of Aβ42 in this concentration range eliciting cellular responses 44 . To address the possibility that DMA's toxicity is affected by the absence or presence of each of these stimulants, we performed the cell viability assays in the absence and presence of LPS and Aβ. Cell viability was evaluated by performing the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The MTT reagent was added to a final concentration of 0.5% and incubated further for 2 h at 37 °C. Subsequently, cell culture media was aspirated and 100 µL of DMSO (BDH) was added to each well to dissolve the formed purple formazan crystals. The absorbance of the resulting purple solution was measured at a wavelength of 570 nm. Three independent experiments were performed in triplicate.
Griess assay. Nitrite (NO −2 ) is one of the primary inert end products of nitric oxide (NO) metabolism in biological systems 42 . NO −2 levels in cell culture supernatants were measured by Griess assay (Promega) to track levels of NO formation indirectly. Samples were prepared by seeding 1.5 × 10 6 SIM-A9 cells in 25 cm 2 flasks and incubating the cells overnight. Cells were then incubated in the absence or presence of DMA (0.1-10 mM) and NF-κB inhibitor BAY 11-7082 (5 μM) (Enzo Lab Sciences) for 2 h. Subsequently, cells were incubated with 1 μg/mL LPS for 24 h. The supernatants were collected after centrifuging at 4 °C and 1000×g for 10 min and then subjected to Griess assay following the manufacturer's instructions. Three independent experiments were performed in triplicate. A standard curve was created as per the manufacturer's instructions to determine the concentration of NO −2 in the samples.
Flow cytometry. Flow cytometry was performed to detect the production of ROS in microglia. To prepare the samples, SIM-A9 cells were seeded at 1.5 × 10 6 cells per 25 cm 2 flasks and incubated at 37 °C overnight. Cells were then incubated in the absence or presence of 10 mM DMA for 2 h followed by 24 h of incubation with 1 μg/ mL LPS. Cells were collected and centrifuged at 10,000 rpm for 5 min. The supernatants were removed, and cells were stained with CellROX Oxidative Stress Reagents (Invitrogen) at 37 °C for 30 min. Samples were then centrifuged at 10,000 rpm for 2 min and washed with phosphate buffered saline (PBS) three times. Cellular fluorescent intensity was detected by BD Accuri C6 flow cytometer (BD Biosciences). Quantification of ROS expression was based on three independent experiments. www.nature.com/scientificreports/ In-vitro assays for secreted cytokines and chemokines. HMC3 cells and SIM-A9 cells were incubated in the absence or presence of DMA (0.1-10 mM) or BAY 11-7082 (5 μM) for 2 h followed by 24 h of 1 μg/ mL LPS or 3 μM Aβ42 stimulation. Cell culture supernatants were collected and centrifuged at 4 °C and 1000×g for 10 min. Enzyme linked immunosorbent assay (ELISA) kits (Thermo Fisher) were used to determine the concentrations of Interleukin (IL)-6, IL-8, IL-1β, tumor necrosis factor (TNF)-α, chemokine (C-C motif) ligand 2 (CCL2), granulocyte-macrophage colony stimulating factor (GM-CSF) and IL-10 following the manufacturer's instructions. Three independent experiments were conducted in duplicate. A second-order polynomial equation generated from a standard curve was used to determine the concentration of each cytokine/chemokine with GraphPad Prism 7 software, as instructed by the manufacturer.
Quantification of Aβ42. Aβ42 levels in microglia cells were evaluated using ELISA (Invitrogen). To prepare the samples, cells were first incubated in the absence or presence of DMA (0.1-10 mM) for 2 h and then incubated with 1 μg/mL LPS for 24 h. Subsequently, cells were collected and lysed in radio immunoprecipitation assay (RIPA) lysis buffer (G-biosciences) with a protease and phosphatase inhibitor cocktail (Thermo Fisher) for 30 min on ice. After centrifugation at 4 °C and 14,000×g for 20 min, the supernatants were collected for further use. The Pierce Bicinchoninic Acid (BCA) Protein Assay Kit (Thermo Fisher) was used to quantify the protein in each sample. Equal amounts of protein were used to conduct the ELISA based on the manufacturer's instructions. Three independent experiments were performed.
Reverse transcription-polymerase chain reaction (RT-PCR). Total RNA was extracted by using the RNeasy Mini kit (Qiagen) and quantified using a Pico200 Microliter UV/Vis Spectrophotometer (VWR). The pure RNA was reverse transcribed to cDNA with SuperScript II Reverse Transcription Kit (Invitrogen). The cDNA samples were further used in Reverse Transcription Polymerase Chain Reaction (RT-PCR) (Agilent Technologies). The 2 −ΔΔCq method was used to quantify gene expression and the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the internal control 45 . The primer sequences used in this analysis are listed in Supplementary Table S1.
Western blot assay. Cells were lysed in RIPA lysis buffer with a protease and phosphatase inhibitor cocktail over the course of 30 min on ice. Samples were then centrifuged at 4 °C and 14,000×g for 20 min. The supernatants were collected, and the protein concentrations were quantified using the BCA Protein Assay Kit. Equal amounts of total protein (30 µg) were resolved by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred onto polyvinylidene fluoride (PVDF) membranes. To conserve reagents, membranes were then cropped and portions of the membrane containing the proteins of interest were used for western blotting. The membranes were blocked with 5% non-fat milk at room temperature for 2 h followed by overnight incubation at 4 °C with primary antibodies (Supplementary Table S2) (Cell Signaling Technologies). The membranes were then incubated at room temperature for 2 h with a 1:1000 dilution of horseradish peroxidase (HRP)-linked secondary antibody (Cell Signaling Technologies) in 5% non-fat milk. The blots were developed using the Pierce enhanced chemiluminescence (Thermo Fisher) and normalized using GAPDH. Quantification of immunoblot was conducted by ImageJ software and based on three independent experiments. Lactate dehydrogenase (LDH) assay. Because the hippocampus comprises a heterogenous population of cells, it is not suited for MTT assay. Therefore, a Pierce lactate dehydrogenase (LDH) assay kit (Thermo Fisher) was used to evaluate the viability of hippocampal slices by measuring the amount of LDH released into the culture medium following an established protocol 46 . After incubating the slices in 50% MEM, 25% Hanks Balanced Salt Solution (with calcium and magnesium) and 25% horse serum in the absence or presence of 1 µg/ ml LPS or 3 μM Aβ42 and 10 mM DMA, the culture medium was collected and centrifuged at 4 °C and 1000×g for 10 min just before the slices were homogenized. The supernatants were then used in an LDH assay according to the manufacturer's instructions. Briefly, 50 μl of each supernatant were added in duplicate into a 96-well plate followed by mixing with 50 μl of Reaction Mixture. The reaction was run for 30 min at 37 °C and the stop solution was then added. The absorbance was measured at a wavelength of 490 nm. Three independent experiments were performed.

Immunofluorescence (IF
Ex vivo assays for secreted cytokines and chemokines. All  www.nature.com/scientificreports/ hippocampi were carefully dissected out and placed in ice cold calcium and magnesium free Hanks balanced salt solution. Hippocampi were then placed on top of a 1.5% agar block sliced at a thickness of 200 microns using a Syskiyou Tissue Slicer (SKU 14240000E). Slices were manually separated and individually immersed in ice cold calcium and magnesium free Hanks balanced salt solution. Using a trimmed transfer pipette, slices were removed from the ice cold media and placed into 6-well plates containing 50% MEM, 25% Hanks Balanced Salt Solution (with calcium and magnesium) and 25% horse serum, pre-warmed to 37 °C. Excess calcium free and magnesium free media on the top of the membrane was removed. Before any treatments with LPS, Aβ42 or DMA, the tissue was equilibrated to 37 °C in a cell culture incubator for 1 h. After the 1-h equilibration, the slices were incubated in the media with or without 10 mM DMA for two hours. Hippocampal slices were then stimulated with either 1 µg/ml LPS or 3 μM Aβ42 for 15 h. Slices were then homogenized in 0.6 mL ice-cold lysis buffer with a protease and phosphatase inhibitor cocktail using a Polytron homogenizer (VWR). The samples were homogenized for 2 min every 15 min over the course of 2 h following published procedures 36,47 . Thereafter, the homogenates were centrifuged at 4 °C and 10,000×g for 15 min, and the supernatants were collected. The BCA assay was used to quantify the protein concentration in the supernatant of each sample. Equal amounts of protein samples were used to perform the ELISA. Three independent experiments (from three different rats) were conducted in duplicate.

Statistical analysis.
All the data were analyzed using GraphPad Prism 7 software and the statistical significance of the differences among and between treatments was tested with one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison post hoc test. A P value of < 0.05 was considered significant and noted based on the following hierarchy *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.

Results
Cell viability by MTT assay. The viability of SIM-A9 and HMC3 cells treated with DMA was assessed by MTT assay (Supplementary Fig. S1). Increasing concentrations of DMA were used to determine the highest non-cytotoxic concentration of DMA in the presence or absence of 1 µg/ml LPS or 3 μM Aβ42. The highest concentration causing less than 20% cell death in SIM-A9 cells was 10 mM. Any change in cell viability at this concentration of DMA was not statistically significant. As for HMC3 cells, the highest concentration of DMA causing less than 20% cell death was 20 mM in unstimulated cells and cells induced by Aβ42 and 30 mM in LPSstimulated cells. There was no significant change in cell viability at this concentration of DMA. To be consistent with the results in SIM-A9 cells, 10 mM DMA was the highest concentration used in both microglial cell lines in all further experiments.

DMA inhibits secretion of NO in SIM-A9 cells. NO is an important mediator produced by inflamma-
tory cells in response to inflammatory stimuli through the induction of inducible nitric oxide synthase (iNOS) 48 . NO -2 levels, an indirect measure of NO production, were significantly increased in SIM-A9 cell supernatants after incubation with 1 µg/ml LPS for 24 h based on a time-course study performed prior to the experiment (Fig. 1A).
In the experiment to test DMA's effect on NO production, 1 µg/ml LPS significantly increased the secretion of NO (reflected by NO -2 measurements) from SIM-A9 cells as compared to untreated cells, as expected (P < 0.001) (Fig. 1B). DMA at a concentration of 10 mM significantly inhibited the secretion of NO (reflected by decreased NO −2 levels ) compared to the LPS control (P < 0.05). The NF-κB inhibitor BAY 11-7082 (5 µM), used as a positive control, had a similar effect (P < 0.05).
Next, the effect of DMA treatment on LPS-induced expression of iNOS in whole cell lysates of SIM-A9 cells was investigated. As expected, there was a significant increase in iNOS expression after a 24 h incubation with 1 µg/ml LPS (P < 0.0001) (Fig. 1C). DMA at 10 mM significantly diminished iNOS production (P < 0.001).

DMA regulates ROS production in SIM-A9 cells. ROS serve as critical inflammatory mediators in
neuroinflammation and have been implicated in the pathogenesis of AD 8 . Flow cytometry was used to investigate the effect of DMA on ROS production (Fig. 2). ROS were detected in 1.17% of unstimulated SIM-A9 cells (Fig. 2). Not surprisingly, 1 µg/ml LPS significantly increased the production of ROS to 9.57% as compared to the control (P < 0.001) (Fig. 2). DMA at a concentration of 10 mM significantly decreased ROS production to 1.30% when compared to the LPS control (P < 0.001) (Fig. 2).

DMA inhibits degradation of IκBα and nuclear translocation of NF-κB p65 in SIM-A9 and HMC3 cells. We have previously shown that DMA prevents activation of the NF-κB pathway by inhibit-
ing IκBα degradation and nuclear translocation of the p65 subunit of NF-κB in LPS-stimulated RAW 264.7 mouse macrophage cells 29,36 . Therefore, we hypothesized that DMA would have the same mechanism of action in microglial cells. IκBα was degraded 15 min after exposure to 1 µg/ml LPS in SIM-A9 cells (Fig. 6A) and HMC3 cells (Fig. 6B) as compared to untreated cells (P < 0.001). DMA prevented LPS-induced IκBα degradation in a concentration-dependent manner, with both 1 mM and 10 mM DMA significantly increasing IκBα levels as compared to the LPS control in SIM-A9 cells (P < 0.05, P < 0.01, respectively, Fig. 6A) and 10 mM DMA significantly increasing IκBα levels as compared to the LPS control in HMC3 cells (P < 0.001, Fig. 6B). DMA's effect on nuclear translocation of the p65 unit of NF-κB in LPS-stimulated microglial cells was then tested. In Fig. 7, SIM-A9 cells were incubated with anti-NF-κB p65 antibodies tagged with green fluorescence and a DAPI stain was used to display the nuclei in blue. Immunoreactivity for the p65 unit in unstimulated SIM-A9 cells was localized primarily in the cytoplasm, as indicated by the green fluorescence surrounding but not overlapping the nucleus (blue) (Fig. 7, first row). However, a 15-min exposure to LPS led to translocation of the NF-κB p65 subunit into the nucleus, reflected by overlap of the green and blue stains (Fig. 7, second row). LPS-stimulated cells treated with 10 mM DMA displayed significantly reduced nuclear staining for p65 compared to the LPS control, consistent with DMA's ability to inhibit LPS-induced nuclear translocation of NF-κB (Fig. 7, third row).
DMA's effect on NF-κB nuclear translocation in Aβ42-stimulated cells was also tested. As expected, immunoreactivity for the p65 unit in untreated SIM-A9 cells was localized primarily in the cytoplasm (Fig. 9). However, a 1 h exposure to 3 mM Aβ42 led to translocation of NF-κB into the nucleus, reflected by overlapping green and blue stains. Aβ42-induced cells treated with 10 mM DMA displayed significantly reduced nuclear staining in comparison with the Aβ42 control, consistent with DMA's ability to prevent Aβ42-induced nuclear translocation of NF-κB p65 (Fig. 9).

DMA does not affect levels of mitogen-activated protein kinase (MAPK) signaling pathway proteins in SIM-A9 cells. The expression of native and phosphorylated forms of JNK, ERK1/2 and p38
in SIM-A9 cells in the absence or presence of stimulation with LPS (1 µg/ml) for 15 min and varying concentrations of DMA were evaluated by immunoblotting (Fig. 10). Phosphorylated JNK, ERK1/2 and p38 MAPK (p-JNK, p-ERK1/2 and p-p38 MAPK) were increased with LPS stimulation (Fig. 10, Lane 2). However, DMA (0.1-10 mM) did not significantly change expression levels of p-JNK, p-ERK1/2 or p-p38 MAPK as compared DMA inhibits secretion of pro-inflammatory cytokines and chemokines from homogenized rat hippocampal slices. Ex vivo cultures provide a platform to study mixed populations of cells while maintaining the three-dimensional structure of the tissue. The hippocampus was chosen because memory loss is a prominent feature of neurodegenerative disorders. Based on LDH assay, 10 mM DMA produced no significant loss of hippocampal cell viability in the absence or presence of LPS ( Supplementary Fig. S2A) or Aβ42 (Supplementary Fig. S2B). Thus, 10 mM DMA was used in ex-vivo experiments. LPS (1 µg/ml) significantly increased the production of IL-10, GM-CSF, CCL2, IL-6 and TNF-α (Fig. 11A-D) and produced a non-significant increase in IL-1β (Fig. 11E) in hippocampal explants when compared to untreated controls. DMA (10 mM) significantly decreased IL-10, GM-CSF and CCL2 levels as compared to LPS controls ( Fig. 11A-C). DMA (10 mM) also inhibited the secretion of IL-6, TNF-α and IL-1β; however, no significant differences were found (Fig. 11D-F). Aβ42 (3 μM) significantly increased the production of IL-6, IL-1β, IL-10 and GM-CSF (Fig. 12A-D) and produced a non-significant increase in the production of TNF-α (Fig. 12E). DMA (10 mM) significantly inhibited the production of IL-6, IL-1β and IL-10 as compared to the LPS controls ( Fig. 12A-C) and reduced GM-CSF and TNF-α levels, although not significantly (Fig. 12D,E).

Discussion
AD is the most common form of dementia 49 , afflicting over 6 million Americans in 2020 and predicted to affect 14 million people by 2060 due to aging of the baby boomer generation and increasing members of the oldest old 50,51 . Despite over a century of investigation, its pathogenesis remains incompletely understood 52 . Currently, the FDA has approved four prescription drugs (donepezil, galantamine, rivastigmine and memantine) to treat the symptoms of different stages of AD. These drugs, however, only provide modest improvement of certain symptoms for limited times and are unable to arrest the progression of AD 53,54 . On June 7 th , 2021, The FDA approved the first Aβ human monoclonal antibody, Aducanumab (marketed as Aduhelm), which was introduced    8,56,57 .
In this work we recognize the importance of neuroinflammation and oxidative stress in the complex array of brain dysfunction leading to AD. In our hands, neuroinflammation induced by LPS in vitro and ex vivo triggers aberrant microglial activation causing increased production of inflammatory factors, such as ROS, NO, pro-inflammatory cytokines and chemokines. ROS itself is mainly generated in mitochondria as a metabolic byproduct, beneficial for some cellular processes, including activation of transcription factors, protein phosphorylation, cell signaling, apoptosis and immunity 58 . However, increased production of ROS induced by LPS stimulates excessive generation of free radicals, resulting in the imbalance between free radicals and antioxidant defenses which is termed oxidative stress 59 . Oxidative stress is detrimental to cellular structures, causes DNA damage and    [60][61][62][63] . Oxidative stress is also implicated in the pathogenesis of AD, based on the finding of abnormal oxidative stress in AD brains and cerebrospinal fluid 64 . Moreover, a myriad of studies have shown that oxidative stress is involved in Aβ accumulation 65,66 . We report here that DMA significantly diminishes LPS-induced ROS expression in SIM-A9 cells.
Besides functioning as a crucial inflammatory mediator, NO is a well-known free radical in intracellular signaling cascades and affects neuronal activity 67 . NO reacts with another free radical, superoxide anion, to produce peroxynitrite, which is toxic to cells and participates in chronic inflammatory and neurodegenerative  www.nature.com/scientificreports/ functions 67,68 . In our SIM-A9 cells, NO is produced through the induction of iNOS which is triggered by activation of the NF-κB pathway 25 . As expected, not only did it increase NO secretion, but LPS also upregulated the expression of iNOS. DMA (10 mM) significantly downregulated both levels of NO and expression of iNOS. LPS significantly stimulates the secretion of IL-6, IL-8, IL-1β, TNF-α, CCL2, GM-CSF and IL-10 in both microglia and hippocampal slices. In particular, IL-6, IL-1β and TNF-α are vital pro-inflammatory cytokines involved in the accumulation of Aβ in AD. High levels of these cytokines inhibit the phagocytosis and clearance of Aβ by microglia and lead to increased amounts of Aβ by promoting cleavage of APP in AD brains [33][34][35]69,70 . It is interesting that IL-10 is up-regulated by LPS and Aβ in cultured microglial and in hippocampal organotypic slices, as IL-10 has both pro-inflammatory and anti-inflammatory properties 71 . DMA significantly attenuated the production of pro-inflammatory cytokines and chemokines induced by LPS in both microglia and hippocampal slices. Furthermore, to mimic the milieu of the AD brain, we stimulated the microglia and hippocampal slices with exogenous Aβ, which resulted in secretion of pro-inflammatory cytokines and chemokines (IL-6, IL-8, IL-1β, TNF-α, CCL2, GM-CSF and IL-10) in both the in-vitro and ex-vivo models. DMA significantly inhibited the generation of these inflammatory mediators. Taken together with the ROS and NO results, we conclude that DMA is a potent anti-neuroinflammatory agent.
There are at least two signaling cascades that may be involved in neuroinflammation: the NF-κB and MAPK signaling pathways 72 . Previously, our laboratory reported that DMA inhibited inflammatory responses via inhibition of the NF-κB signaling pathway but had no effect on the MAPK pathway 29 . Consistent with our previous work, we report here that DMA inhibited the degradation of IκBα and translocation of NF-κB p65 into the nucleus in stimulated cells but had no effect on the MAPK pathway. Besides its effect on neuroinflammation, we also report here that DMA significantly downregulated the gene and protein expression of APP, further contributing to the decreased expression of Aβ in microglia. Interestingly, DMA did not affect levels of tau protein (data not shown).
We recognize that many factors affecting the efficacy of DMA in the CNS may not be recapitulated in our ex-vivo and in-vitro models. For example, stimulation of microglia and hippocampal slices with both LPS and Aβ increased IL-10 levels, whereas IL-10 has anti-inflammatory function in many in-vivo systems. Pre-clinical trials to test DMA's efficacy in in-vivo models of neuroinflammation and/or AD are planned.

Conclusion
To the best of our knowledge, the present study is the first report of DMA's effect on neuroinflammation and APP production. A summary of DMA's proposed mechanism of action is provided in Fig. 13. Briefly, stimulated pattern recognition receptors in microglia activate the NF-κB pathway, leading to degradation of IκBα